PROJECT SUMMARY Under standard culture conditions, the profile of the secondary metabolites that fungi are biosynthesizing is limited to the carbon sources that are in the media. Genomic data from fungi indicate that numerous biosynthetic gene clusters are silent, and thus, the true chemical potential of a fungal culture is perhaps unknown, or at least, unobserved. While fungi have a rich history in providing drug leads (such as penicillin), the discovery of new leads is likely incumbent upon discovering new bioactive molecules. By taking advantage of the fact that fungi have evolved ways to survive in complex environments, as well as to respond chemically to different environmental cues, co-culturing is a way to activate the untapped biosynthetic potential of fungi. The droplet-probe will be used to analyze, in situ, the chemical profiles of secondary metabolites to obtain a spatial map of how fungi distribute their secondary metabolites during these chemical interactions. Genomics will be used to link the secondary metabolites produced in co-culture to the producer strain. This study will triangulate information from biology/mycology (fungal co-culture), metabolomics and genomics (to locate producer species), and natural products chemistry (isolation and elucidation of new chemistry) to uncover and activate silent gene clusters to search for unique secondary metabolites. Preliminary data demonstrate that upon co-culture of fungi, the biosynthesis of secondary metabolites can be stimulated. This is likely because they interact chemically, essentially fighting for their resources. Xylaria cubensis, a strain that produces the FDA approved fungistatic drug griseofulvin, will be used as a model organism for co-culturing. Other endophytic strains, such as Aspergillus sp. and Penicillium sp., will be used along side X. cubensis in the co- culture experiments, because they produce iconic molecules. In addition, we will target genetically tractable organisms, since genome studies have shown that members of these genera have rich biosynthetic gene clusters that produce unknown chemical entities. The mono and co-cultures will undergo untargeted metabolomics, and PCA will be used to analyze the chemical differences between the mono and co-cultures. For training, the applicant is part of a natural products chemistry research group as a T32 Fellow in the Department of Chemistry & Biochemistry at the University of North Carolina at Greensboro. Her skills in that regard will be further honed via the conduct of Aims 1 and 2. Additionally, so as to further her training, and to prepare her for a future where genomics will become an even great part of natural products research, she will receive hands-on training in fungal genomics as part of Aim 3 via close interaction with the research group of Dr. Antonis Rokas at Vanderbilt University. Finally, to complement her ability to communicate research, she will receive training in scientific writing, which is currently an underdeveloped skill set of the applicant.